Endorepellin: methods and compositions for inhibiting angiogenesis

ABSTRACT

Angiogenesis is a highly regulated process, yet many diseases are driven by unregulated angiogenesis. For example, the growth and metastasis of tumors is dependent on the growth of new blood vessels, which nurture the tumor by providing not only a growth-conducive environment but also a route by which metastatic cells can escape. The present invention provides an angiogenesis inhibiting agent endorepellin, its fragments or derivatives, or analogs thereof, which inhibit angiogenesis. The invention disclosed herein provides a novel therapeutic approach to the treatment of tumors, and other angiogenesis-mediated diseases or conditions, by inhibiting the generation of new blood vessels. The present invention also provides anti-endorepellin, its fragments or derivatives, or analogs thereof, antibodies for the detection, diagnosis and monitoring of angiogenesis-mediated diseases or conditions.

GOVERNMENT RIGHTS IN THE INVENTION

This invention was made with government support under grant CA47282 awarded by the NIH.

FIELD OF THE INVENTION

The present invention relates to the fields of tumor biology and molecular biology, and to a method of treating angiogenesis-related diseases or conditions and, more particularly, to the inhibition of neovascularization and tumor growth and metastasis in vivo.

BACKGROUND OF THE INVENTION

Perlecan is a modular proteoglycan that participates in the formation and maintenance of basement membranes in various organs (1,2). It is a major heparan sulfate proteoglycan (HSPG) secreted by endothelial cells and is a potent inhibitor of smooth muscle cell proliferation, a biological function mediated by perlecan's block of fibroblast growth factor 2 (FGF2) activity (13) and Oct-1 gene expression (14). Perlecan has been shown to play a critical role in regulating the vascular response to injury in vivo (17).

Angiogenesis is one of the most important events in tumor progression and is greatly influenced by cell-matrix interactions taking place at the surface of the endothelial cells and at the tumor-matrix boundaries (31). HSPGs act as depots for pro- and anti-angiogenic factors and, in concert with members of the FGF and vascular endothelial growth factor (VEGF) families and their receptors, modulate various aspects of angiogenesis (9). Interestingly, various angiogenesis inhibitors have been found to be proteolytically-processed forms of basement membrane collagens types IV, XV and XVIII, the latter two being chondroitin and HSPGs, respectively (32).

The invention presented herein describes a potent inhibitor of angiogenesis: the carboxyl terminus domain, domain V, of perlecan. This fragment, “endorepellin,” inhibits angiogenesis and is active at nanomolar concentrations. Endorepellin interferes with endothelial cells' adhesive properties for various substrata, including, but not limited to, fibronectin and fibrillar collagen. Endorepellin, thus, represents a novel anti-angiogenic tool for the treatment of diseases or conditions associated with movement, migration and adhesion of cells, including diseases that involve angiogenesis such as, but not limited to, tumor metastasis and growth. The present invention provides endorepellin, analogs and fragments thereof, for use in inhibiting angiogenesis.

Abbreviations

“HSPG” means “heparan sulfate proteoglycans”

“HUVEC” means “human umbilical vein endothelial cells”

“CAM” means “chicken chorioallantoic membrane

“FGF” means “fibroblast growth factor”

“VEGF” means “vascular endothelial cell growth factor”

“LG” means “laminin-type G”

“EG” means “epidermal growth factor-like”

Definitions

“patient” as used herein can be one of many different species, including but not limited to, mammalian, bovine, ovine, porcine, equine, rodent and human.

“analog” as used herein is a derivative or modification of the native sequence. One skilled in the art may prepare such analogs wherein the native sequence is modified by resultant single or multiple amino acid substitutions, additions or deletions. All such modifications resulting in a derivative of endorepellin are included within the scope of the invention, provided that the molecule retains angiogenesis-inhibiting activity.

“substantial sequence homology” means at least approximately 60% homology between the amino acid residue sequence in the endorepellin analog or derivative sequence and that of endorepellin sequence.

“anti-angiogenesis activity” and “angiogenesis-inhibiting activity” refers to the ability of a molecule to inhibit the growth of blood vessels.

“angiogenesis-mediated disease” refers to the unregulated growth of new blood vessels that causes a disease or exacerbates an existing condition.

DESCRIPTION OF THE DRAWINGS

FIG. 1. Perlecan domain V (endorepellin) binds to the anti-angiogenic factor endostatin. FIG. 1a, Agarose gel showing the 1.7 kb cDNA strongly interacting with endorepellin, obtained from the BgIII digestion of clone A3. Complete sequence of A3 clone revealed the C terminus of type XVIII collagen. FIG. 1b, Schematic representation of the human α chain of type XVIII collagen. The triple-helical and non-triple helical domains are indicated by rods and blue boxes, respectively. The C-terminal endostatin fragment is highlighted in orange. The beginning of the clone A3 sequence is shown (NCBI accession # AF018082). FIG. 1c, Growth and β-galactosidase activity triggered by the interaction of endorepellin with collagen type XVIII fragment compared to the positive (p53 and T-antigen) and negative control (lamin and T-antigen). FIG. 1d, Co-immunoprecipitation of collagen XVIII (clone A3) and endorepellin following in vitro transcription/translation using [³⁵S]methionine as the labeled precursor. Endorepellin (lane 1) and collagen XVIII (lane 2) are mixed in equimolar amounts and co-immunoprecipitated with either anti-hemagglutinin (α-HA) (lane 3) or no antibody. FIG. 1e, Co-immunoprecipitation of endostatin with endorepellin. Domain III (lane 1), endorepellin (lane 2) and endostatin (lane 3) were generated by in vitro transcription/translation using [³⁵S]methionine as the labeled precursor. Endostatin was mixed with either domain III (lane 4) or endorepellin (lane 5) and immunoprecipitated with anti-hemagglutinin (α-HA) antibody. FIG. 1f, Schematic representation of domain V and various deletion mutants. Orange ovals indicate laminin-type G modules (LG), whereas blue rectangles indicate EGF-like (EG) modules. The growth is indicated by semi-quantitative assessment with maximal growth at +++. The numbers within parentheses designate the amino acid position based on the mature protein core. FIG. 1g, Representative α and β-galactosidase assays of various deletion mutants, as indicated; pGB53/pGADT was the positive control.

FIG. 2. Endorepellin is a powerful anti-angiogenic factor. FIG. 2a, Purification of endorepellin from media conditioned by 293-EBNA cells expressing the 81 kDa endorepellin tagged with His6. Coumassie-stained SDS-PAGE (left) and Western immunoblotting with anti-His6 antibody (right) of negative control media (lanes 1 and 4), flow through (lanes 2 and 5), and 250 mM imidazole eluate (lanes 3 and 6). FIG. 2b and FIG. 2c, HUVEC migration assays through fibrillar collagen using 10 ng/ml VEGF as a chemotactic inducer and preincubation the HUVECs for 30 min with various concentrations of endostatin (ES) and endorepellin (ER). Serum free medium (SFM). FIG. 2d, CAM assays three days after the application of sponges containing VEGF (1 ng), VEGF (1 ng)+endorepellin (400 ng), or buffer alone. Scale bar, 1 mm.

FIG. 3. Endorepellin, but not endostatin, blocks endothelial tube formation induced by fibrillar collagen. FIG. 3a, FIG. 3b, FIG. 3c, and FIG. 3d, Gallery of light micrographs capturing the time course production of HUVEC tube-like formation in fibrillar collagen containing either buffer (Control), endorepellin, endostatin, or both at the designated concentrations. In this assay, 4×10⁵ cells are incubated for 24 hr and pictures are taken at various intervals as indicated in the top margins. Scale bar, 250 μm.

FIG. 4. Biological consequences of endostatin/endorepellin interaction. FIG. 4a and FIG. 4b, HUVEC migration assays through fibrillar collagen using 10 ng/ml VEGF as a chemotactic inducer and preincubation the HUVECs for 30 min with various concentrations of endostatin (ES), endorepellin (ER), or various combinations as indicated. The values are presented as the percentage of maximal stimulation induced by VEGF alone, arbitrarily set at 100%. FIG. 4a is the summary of three independent experiments run in quadruplicates, mean ±SE. The values in FIG. 4b derive from an additional experiment run in quadruplicate, mean ±SE. Serum free medium (SFM).

FIG. 5. Endorepellin is counter-adhesive for endothelial, fibrosarcoma and colon carcinoma cells. FIG. 5a, HUVEC adhesion to increasing concentrations of various substrata including fibronectin (★), BSA (), or endorepellin (θ). For each point, 5×10⁴ cells are seeded on the various substrata. After 1 h, adherent cells are washed, stained with crystal violet and solubilized in 0.1% Triton X-100, and absorbance monitored at 600 nm. The number of attached cells is proportional to the absorbance. About 80% of the total cells are attached in the plateau region of the fibronectin curve. The values represent the mean ±SE (n=4). FIG. 5b, Gallery of light micrographs of crystal violet-stained HUVECs adhered to 50 nM fibronectin following incubation for 1 hr with endorepellin at the indicated concentrations. Scale bar, 125 μm. FIG. 5c and FIG. 5d, Adhesion assays for HT1080 fibrosarcoma and WiDr colon carcinoma cells, respectively, on fibronectin (★), BSA (), or endorepellin (θ) substrata. The conditions are identical to those described in panel a. The values represent the mean ±SE (n=4). FIG. 5e and FIG. 5f, Displacement curves employing increasing concentration of either endorepellin or endostatin, respectively. The calculated IC₅₀ for HT1080 and WiDr was 110 and 40 nM, respectively. The values represent the mean ±SE (n=4).

DESCRIPTION OF THE INVENTION

Angiogenesis, the process by which new blood vessels are formed, is essential for normal bodily activities including reproduction, development and wound repair. Although the process is not completely understood, angiogenesis is believed to involve a complex interplay of molecules, which regulate the growth of endothelial cells (the primary cells of capillary blood vessels). Under normal conditions, these molecules appear to maintain the microvasculature in a quiescent state (i.e. one of no capillary growth) for prolonged periods which may last for as long as weeks or, in some cases, decades. When necessary (such as during wound repair), these same cells can undergo rapid proliferation and turnover within a 5 day period.

Although angiogenesis is a highly regulated process under normal conditions, many diseases (characterized as angiogenic diseases) are driven by persistent unregulated angiogenesis. Otherwise stated, unregulated angiogenesis may either cause a particular disease directly or exascerbate an existing pathological condition. For example, growth and metastasis of solid tumors are dependent on angiogenesis. It has been shown, for example, that tumors which enlarge to greater than 2 mm must obtain their own blood supply and do so by inducing the growth of new capillary blood vessels. Neovascularization of a tumor enables the metastatic spread of tumor cells by providing a route of escape for the metastatic cells and nurturing the tumor by providing a growth-conducive environment. Another example is ocular neovascularization, a disease that has been implicated as the most common cause of blindness and dominates approximately twenty distinct eye diseases. In certain existing conditions, such as arthritis, newly formed capillary blood vessels invade the joints and destroy cartilage. In diabetes, new capillaries formed in the retina invade the vitreous, bleed, and cause blindness.

Many other diseases are driven by persistent unregulated angiogenesis. The diseases or conditions treatable by the present invention include, but are not limit to, primary tumor growth, tumor invasion or metastasis, hemangioma, leukemia, atherosclerosis, post-balloon angioplasty, myocardial angiogeneisis, plaque neovascularization, vascular restenosis, neointima formation following vascular trauma, vascular graft restenosis, fibrosis associated with a chronic inflammatory condition, lung fibrosis, chemotherapy-induced fibrosis, wound healing with scarring and fibrosis, psoriasis, deep venous thrombosis, corneal diseases, ovulation, menstration, placentation, or any other disease or condition which is mediated by angiogenesis. In addition, an important new medical method for birth control, wherein an effective amount of endorepellin is administered to a female such that uterine endometrial vascularization is inhibited and embryo implantation does not occur or is not sustained, is also contemplated by the present invention.

Angiogenesis Inhibitors

Angiogenesis inhibitors currently under development for use in treating angiogenic diseases have many disadvantages. Suramin, for example, is a potent angiogenesis inhibitor but causes severe systemic toxicity in humans at doses required for antitumor activity. Compounds such as retinoids, interferons and anti-estrogens are safe for human use but have weak anti-angiogenic effects. Thus, there is a need for compounds useful in treating angiogenic diseases in mammals. More specifically, there is a need for angiogenesis inhibitors which are safe for therapeutic use and which exhibit selective toxicity with respect to the pathological condition, such as by selectively inhibiting the proliferation of cancer cells while exhibiting no, or a low degree of toxicity to normal (ie. non-cancerous) cells. The present invention provides such a compound, endorepellin, to treat angiogenic-mediated diseases in mammals.

Furthermore, certain types of tumors are more amenable to therapy than others. Such tumors include, soft tissue tumors such as tumors of the blood. The reason for the increased efficacy in treatment regimens with respect to these tumors is their greater accessibility to chemotherapeutic agents. In contrast, it is much more difficult for most chemotherapeutic agents to reach all of the cells of a solid tumor mass.

Solid tumors rely on the generation of new blood vessels in order for the nutrients to reach the cells within the tumor. Thus, the present invention provides a novel therapeutic approach to the treatment of solid tumors wherein the generation of new blood vessels within the tumor, rather than the tumor cells themselves, is the target. This treatment is not likely to lead to the development of resistant tumor cells. Moreover, inhibition of angiogenesis leads to an amplification of the anti-tumor effect since many tumor cells rely on a single vessel for their nutrients.

Methods

Standard techniques are used for recombinant nucleic acid methods, polynucleotide synthesis, cell culture, and transgene incorporation (e.g., electroporation, microinjection, lipofection). The enzymatic reactions, oligonucleotide synthesis, and purification steps are performed according to the manufacturer's specifications. The techniques and procedures are performed according to conventional methods in the art and various general references that are provided throughout this document. The procedures therein are well known in the art, some of which are provided for the convenience of the reader.

Primary Culture and Cell Lines

Primary cultures of HUVECs are prepared from fresh umbilical cords and cultured on collagen-coated flasks in M199 or M200 medium supplemented with 10% FBS, 50 μg/ml heparan and endothelial cell growth supplement, isolated from bovine hypothalami. Only passages 4 to 8 are used. A431 squamous carcinoma cells, HT1080 fibrosarcoma, WiDr colon carcinoma, MCF7 breast carcinoma, and M2 mouse melanoma cells are obtained from American Type Culture Collection (Manassas, Va.).

Yeast Two-hybrid Screening and Co-immunoprecipitation

To reduce the number of false positives, the Matchmaker GAL4 two-hybrid system 3 (Clontech, Palo Alto, Calif.), which adopts three independent reporter genes for the selection, His, Ade and α or β-galactosidase, is used. A keratinocyte library constructed in the pACT2 vector is amplified and the plasmids are extracted employing the NucleoBond AX Giga plasmid purification kit (both the vector and kit are from Clontech). Endorepellin is subcloned into the pGBKT7 vector using PCR generated fragments as previously described (44), and is used as a bait to screen ˜5×10⁶ cDNAs. The yeast clones growing in selective medium are re-plated in quadruple minus plates containing X-gal. The plasmids from the blue colonies are isolated, expanded by subcloning in DH5α bacterial cells, and analyzed on agarose gels after restriction digestion with BgIII. The inserts are identified by automatic sequencing.

Seven deletions of perlecan domain V are obtained by PCR, including suitable restriction sites to allow unidirectional ligation into the pGBKT7 vector (44). The various proteins are in vitro transcribed and translated in presence of [³⁵S]methionine (ICN Pharmaceuticals, Costa Mesa, Calif.) employing the TNT® reticulocyte lysate system (Promega, Madison, Wis.). One μg of the pGBKT7 or pGADT7 constructs is employed and the reactions are incubated for 90 min at 30° C. Aliquots are subjected to co-immunoprecipitation with affinity-purified, anti-hemagglutinin (αHA) rabbit polyclonal antibodies (Clontech). The immune-complexes are captured with protein A/G agarose beads (Pierce, Rockford, Ill.). The beads are washed three times with HNTG buffer (10 mM Hepes (pH 7.4), 150 mM NaCl, 0.1% triton X100, 10% glycerol, 200 mM Na₂VO₄, 20 mM NaF and an EDTA-free protease inhibitors cocktail (Roche Diagnostics GmbH, Mannheim, Germany). The bands are separated in polyacrylamide gels, fixed in the presence of AMPLIFY™ (Amersham Pharmacia Biotech, Uppsala, Sweden), dried under vacuum and exposed to KODAK films.

Expression and Purification of Recombinant Endorepellin

The pCEP-Pu vector bearing the sequence of the BM40 signal peptide is used to transfect by electroporation ˜10⁷ human kidney cells (293-EBNA) expressing the Epstein-Barr virus nuclear antigen (EBNA)-1. Mass cultures are selected in media containing 250 μg/ml G418 and 500 ng/ml puromycin. The confluent cells are allowed to express the recombinant protein in serum free for 48 hr. The conditioned media are concentrated in a dialysis bag with PEG and dialyzed in sonication buffer (12.5 mM Na₃PO₄, 75 mM NaCl, pH=8.0) and further purified employing the Ni-NTA resin and eluted with 250 mM imidazole. The fractions containing the recombinant protein are dialyzed against 10 mM Hepes, 150 mM NaCl and 2 mM EDTA and further concentrated. In all the purification steps phenylmethylsulfonyl fluoride (2 mM) and N-ethylmaleimide (2 mM) are the protease inhibitors. Using this procedure, 5-10 mg of endorepellin/L is routinely purified from conditioned medium. ELISA and immunoblotting with anti-domain V (16) or Penta-His (QIAGEN, Valenica, Calif.) monoclonal antibodies is performed as described before (24).

Endothelial Cell Migration, Tube Formation and Chorioallantoic Membrane (CAM) Assays

A 48-well Boyden chamber (Neuroprobe Inc., Gaithersburg, Md.) is used for migration assays employing HUVECs. VEGF165 (R&D Systems, Minneapolis, Minn.) is used as a chemo-attractant in M199 (Life Technologies, Carlsbad, Calif.) containing 0.1% BSA. Cells migrated through eight-micron nucleopore polyvinylpyrrolidine-free polycarbonate filters (Corning, Cambridge, Mass.) pre-coated for 48 hr with 100 μg/ml collagen type I (Collaborative Biomedical Products, Bedford, Mass.) in 0.2 N acetic acid and air dried. The endothelial cells, trypsinized and re-suspended in M199 medium, are pre-incubated for 1 h with different concentrations of endorepellin and/or endostatin (Calbiochem-Novabiochem, San Diego, Calif.). The cells are allowed to migrate through the filter for 6 h at 37° C. with 5% CO₂. Every experiment is performed in quadruplicate, after each incubation, the filters are washed, fixed, stained with Diff-Quick stain (VWR Scientific Products, Bridgeport, N.J.), and counted in total using conventional microscopy.

For in vitro tube-like formation, 12-well cell culture dishes are coated with 100 μg/ml collagen type I in sterile 10 mM acetic acid. HUVECs seeded for 18 hr, and then covered with a second layer of collagen (41). Cultures are incubated until gels had solidified, typically 15-30 min, and then given 1 ml of media containing the various test agents and control substances.

For the CAM assays, fertilized White Leghorn chick eggs are incubated at 37° C. After three days of incubation, ˜3 ml of albumin was removed to detach the CAM. A small square window is then opened in the eggshell, which is sealed with tape and the eggs are returned to the incubator. At day 9, a ˜1 mm³ Gelfoam sterile sponge Gelfoam (Pharmacia, North Peapak, N.J.) is placed on the chorioallantoic membranes and various test factors are applied including VEGF, endorepellin or buffer alone. The development of blood vessels is monitored by light microscopy up to 12 days.

Cell Adhesive and Counter-adhesive Assays

HUVECs and various tumor cells lines, including WiDr, A431, MCF7 and M2 cells, are tested for adhesion to various substrata including fibronectin, collagen type I, BSA, endorepellin or endostatin as plastic-immobilized substrata using dose-response profiles (coating concentrations of 10-180 nM). 5×10⁵ cells are plated in quadruplicate wells and, after 1 hr of incubation, adherent cells are washed, fixed in 1% glutaraldehyde for 10 min, stained with crystal violet, lysed with 0.2% Triton X-100, and assayed by a colorimetric test (33). The crystal violet concentration, which is proportional to the cell number, is estimated at A₆₀₀.

The anti-adhesive assays are performed in a similar way. About 40 μg/ml fibronectin are used to coat individual plates. After blockage with 1% BSA, the cells are added to the wells in the presence of increasing concentration of endorepellin or endostatin. After 1 h of incubation, the wells are treated as above. Additional details are provided in the legends to the figures and in the methods (supra).

Endorepellin, Analogs and Fragements Thereof

Thus, the present invention contemplates amino acid residue sequences that have substantial sequence homology to endorepellin such that those sequences demonstrate like biological activity. It is well known in the art that modifications and changes can be made to a peptide without substantially altering the biological function of that peptide. For example, alterations to endorepellin peptide fragments may enhance the peptide's potency or stability to enzymatic breakdown. Such contemplated sequences include those analogous sequences characterized by a change in amino acid residue sequence or type wherein the change does not alter the fundamental nature and biological activity of the aforementioned endorepellin.

Generation of Antibodies to Endorepellin and Uses Thereof

According to the invention, endorepellin, its fragments or derivatives, or analogs thereof, may be used as an immunogen to generate antibodies that recognize such an immunogen. Such antibodies include but are not limited to polyclonal, monoclonal, chimeric, single chain, Fab fragments, and Fab expression library. In a specific embodiment, antibodies to endorepellin are produced.

Methods of producing endorepellin for generation of antibodies include, but are not limited to, recombinant DNA techniques, peptide synthesis wherein multiple fragments are synthesized and subsequently linked together to form the full length endorepellin, or proteolyic digestion of perlecan. These protocols are standard technology and well known to those of skill in the art.

Various procedures known in the art may be used for the production of polyclonal antibodies to endorepellin or derivatives or analogs thereof. For the production of antibody, various host animals can be immunized by injection with the native endorepellin, or a synthetic version, or derivative (e.g., fragment) thereof, including but not limited to rabbits, mice, rats, etc. Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum.

For preparation of monoclonal antibodies directed toward an endorepellin sequence or analog thereof, any technique that provides for the production of antibody molecules by continuous cell lines in culture may be used. Examples of such techniques include, but are not limited to, the hybridoma, as well as the trioma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique to produce human monoclonal antibodies. In an additional embodiment of the invention, monoclonal antibodies can be produced in germ-free animals utilizing recent technology.

In the production of antibodies, screening for the desired antibody can be accomplished by techniques known in the art, such as, but not limited to, ELISA (enzyme-linked immunosorbent assay). For example, to select antibodies that recognize a specific domain of endorepellin, one may assay generated hybridomas for a product that binds to an endorepellin fragment containing such domain.

Diagnostic Uses of Antibodies

Anti-endorepellin antibodies may be generated (supra) and used to detect, prognose, diagnose, or monitor the treatment of angiogenesis-mediated diseases, such as cancer, by detecting the presence of endorepellin in patient samples. For detection of endorepellin sequences, a diagnostic kit of the present invention comprises, in one or more containers, an anti-endorepellin antibody that can be detectably labeled. In a different embodiment, the kit can comprise, in one or more containers, a labeled specific binding portion of an antibody. As used herein, the term “detectable label” refers to any label that provides directly or indirectly a detectable signal and includes, for example, enzymes, radiolabelled molecules, fluorescent molecules, particles, chemiluminesors, enzyme substrates or cofactors, enzyme inhibitors, or magnetic particles. Examples of enzymes useful as detectable labels in the present invention include, but are not limited to, alkaline phosphatase and horse-radish peroxidase. A variety of methods are available for linking the detectable labels to antibodies and include for example the use of a bifunctional agent, such as, 4,4′-difluoro-3,3′-dinitro-phenylsulfone, for attaching an enzyme, for example, horse radish peroxidase, to an antibody. The attached enzyme is then allowed to react with a substrate yielding a reaction product that is detectable. The present invention provides a method for detecting endorepellin in a patient sample, comprising, contacting the patient sample with an anti-endorepellin antibody under conditions such that immunospecific binding occurs, and detecting or measuring the amount of any immunospecific binding by the antibody.

Patient samples are any sample from a patient thought to contain endorepellin. Samples include, but are not limited to, peripheral blood or serum, urine, tissue sections, peritoneal fluid, cerebrospinal fluid, uterine fluid, mucus, etc.

Administration

This invention includes methods for inhibiting angiogenesis. By inhibiting angiogenesis, tumor metastasis is inhibited. In this method, a patient is administered an amount of endorepellin, its fragments or derivatives, or analogs thereof, effective to inhibit angiogenesis. The compound, or pharmaceutically acceptable salt thereof, is administered in the form of a pharmaceutical composition (infra).

Doses of the compounds include pharmaceutical dosage units comprising an effective amount of the peptide. By an effective amount is meant an amount sufficient to achieve a steady state concentration in vivo which results in a measurable reduction in any relevant parameter of disease, such as growth of primary or metastatic tumor, any accepted index of inflammatory reactivity, or a measurable prolongation of disease-free interval or survival.

In one embodiment, an effective dose is approximately between 10-fold and 100-fold higher than the 50% inhibitory concentration (IC₅₀) of the compound.

The amount of active compound to be administered depends on the precise peptide or derivative selected; the disease or condition; the route of administration; the health and weight of the recipient; the existence of other concurrent treatment; if any, the frequency of treatment, the nature of the effect desired, for example, inhibition of tumor metastasis; and the judgment of the skilled practitioner. The precise dose to be employed is decided according to the judgement of the practitioner and each patient's circumstances. The following are examples of such doses and is not meant to limit the invention in any way.

The proteins and protein fragments with endorepelliin activity are provided as isolated and substantially purified proteins and protein fragments in pharmaceutically acceptable formulations using formulation methods known to those of ordinary skill in the art. These formulations are administered by standard routes. In general, the combinations may be administered by the topical, transdermal, intraperitoneal, intracranial, intracerebroventricular, intracerebral, intravaginal, intrauterine, oral, rectal or parenteral (e.g., intravenous, intraspinal, subcutaneous or intramuscular) route. In addition, the endorepellin, its fragments or derivatives, or analogs thereof, may be incorporated into biodegradable polymers allowing for sustained release of the compound, the polymers being implanted in the vicinity of where drug delivery is desired, for example, at the site of a tumor or implanted so that the endorepellin, its fragments, derivatives, or analogs thereof, is slowly released systemically. Osmotic minipumps also may be used to provide controlled delivery of high concentrations of endorepellin, its fragments, derivatives, or analogs thereof, through cannulae to the site of interest, such as directly into a metastatic growth or into the vascular supply to that tumor.

An exemplary dose for treating a patient with a tumor is an amount of up to about 100 milligrams of active compound per kilogram of body weight.

Typical single dosages of the peptide are between about 1 μg and about 100 mg/kg body weight. For topical administration, dosages are in the range of about 0.01-20% concentration of endorepellin, its fragments or derivatives, or analogs thereof. A total daily dosage in the range of about 10 milligrams to about 7 grams is exemplary for oral administration. The foregoing ranges are, however, exemplary, as the number of variables in regard to an individual treatment regimen is large, and considerable excursions from these recommended values are expected.

The therapeutic regimen with endorepellin, its fragments or derivatives, or analogs thereof, compounds of the invention produce an inhibitory effect on cell migration and invasion, on angiogenesis, on tumor metastasis and/or on inflammatory reactions in a patient having a disease or condition associated with undesired cell invasion, migration-induced proliferation, angiogenesis or metastasis (supra).

Endorepellin, its fragments, derivatives, or analogs thereof, of the present invention are useful for inhibiting angiogenesis when used alone or in combination with other compositions and procedures for the treatment of diseases. For example, a tumor may be treated conventionally with surgery, radiation or chemotherapy and endorepellin, or analogs or derivatives thereof, administration, either concurrently or subsequently, to extend the dormancy of micrometastases and to stabilize and inhibit the growth of any residual primary tumor.

Pharmaceutical Compositions

The present invention also provides pharmaceutical compositions. Such compositions comprise a therapeutically effective amount of endorepellin, its fragments, derivatives, or analogs thereof, and a pharmaceutically acceptable carrier or excipient. Such a carrier includes, but is not limited, to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The carrier and composition are sterile. The formulation suits the mode of administration.

The composition, if desired, also can contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc.

In one embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition also may include a solubilizing agent and a local anesthetic such as lidocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

The endorepellin, its fragments, derivatives, or analogs thereof, of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

Results

Endostatin is a Novel Interacting Partner for Perlecan Domain V/Endorepellin

Using endorepellin as bait, a keratinocyte cDNA library is screened in the yeast two-hybrid system. One of the strongest interacting clones (clone A3) encoded the C-terminal portion of collagen type XVIII, containing the NC1 domain and the potent anti-angiogenic factor endostatin (FIGS. 1a and b). Since endostatin inhibits endothelial cell proliferation and effectively arrests the growth of several tumors (28), and because perlecan and endostatin co-localize in most tissues (29, 30), it is reasoned that an interaction between these two proteins could occur in vivo and could play a role in tumor progression. Therefore, the collagen fragment is sub-cloned into the pGADT7 vector, and the interaction with endorepellin is once more tested with the two-hybrid system on a one-to-one basis. The growth of the cells in quadruple minus medium is comparable to that of the positive control (pGBKT7-53/pGADT7-T), as well as the blue color generated by α-galactosidase expression (FIG. 1c).

To corroborate the yeast interaction, collagen XVIII and endorepellin are transcribed and translated in vitro, resulting in the ˜81 and ˜65 kDa fragments, respectively (FIG. 1d). The two proteins are co-precipitated with an anti-hemagglunitin antibody which recognizes the oligopeptide epitope HA present at the C-terminus of collagen XVIII (FIG. 1d). To map the site of interaction, a deletion fragment of NC1 domain is cloned into pGADT7 containing only endostatin. The NC1 domain is then transcribed in vitro and translated resulting in a 23 kDa band (FIG. 1e, lane 3). As a further control, perlecan domain III is transcribed and translated, producing a ˜130 kDa band (FIG. 1e, lane 1). The results show that only endorepellin interacts with endostatin (FIG. 1e, lane 5). In contrast, domain III does not bind to endostatin (lane 4). These experiments are repeated three times with comparable results.

Endostatin Interacts Specifically with the LG2 Module of Endorepellin

To establish the precise location of this interaction, seven deletion mutants of the domain V/endorepellin (SEQ ID NO:3) fragment of perlecan (SEQ ID NO:2) were generated, Δ1-Δ7 (SEQ ID NOS:4, 5, 6, 7, 8, 9, and 10) (FIG. 1f). This domain consists of three laminin type G (LG1-LG3) modules separated by four EGF-like (EG1-EG4) modules in an arrangement highly conserved across species (1). Robust growth in quadruple minus media is observed in cells co-transformed with full-length endorepellin (SEQ ID NO:3) and endorepellin with two deletions, Δ1 (SEQ ID NO:4) and Δ5 (SEQ ID NO:8), which contain the LG2 module (FIG. 1f). These results are corroborated by α- and β-galactosidase assays (FIG. 1g). Further support for a true protein/protein interaction is growth in amino acid-deficient media, transcription of LacZ (α- and β-galactosidase) under the control of distinct GAL4 upstream activating sequences, and the subsequent ability of the co-transformant yeast strains to express functional galactosidase activity. Thus, the LG2 module of endorepellin is the specific site of endostatin binding.

Recombinant Endorepellin is Anti-angiogenic

Human recombinant endorepellin, generated in 293-EBNA cells, migrated on SDS-PAGE as a single band of the predicted ˜81 kDa. The identity of this recombinant protein is further confirmed by immunoblotting with anti-His6 antibody (FIG. 2a) and ELISA, using a specific monoclonal antibody against domain V (12). To test the biological properties of endorepellin, VEGF-induced migration of human umbilical vein endothelial cells (HUVEC) subcloned to passages 4-8 (31) is used. It is well established that the motility and vectorial migration of endothelial cells that occur with invasion is a fundamental component of angiogenesis (27,32). When VEGF is used in the lower chamber, there is a complete suppression of HUVEC migration through the membrane at 1-10 μg/ml (12-120 nM) endorepellin (FIG. 2b). Interestingly, endorepellin is more active than recombinant endostatin purified from Pichia pastoris yeast cells. Subsequent dilution experiments revealed that endorepellin is fully active at 0.5 μg/ml (6 nM) (FIG. 2c), with a calculated IC₅₀ of 1.2 nM (±0.1, n=11). In some preparations, endorepellin is active even at picomolar concentrations, similar to those reported for endostatin produced by 293-EBNA cells (31), the same cells used in this study. The experiments in FIGS. 2b and c are repeated eleven times with various preparations of endorepellin, and a marked suppression of HUVEC migration is consistently found. In contrast to endostatin, the migratory response is not dependent on preincubation of the endothelial cells with endorepellin. In experiments where endorepellin is placed in the lower chambers of the invasion assay, similar inhibition of VEGF-induced migration is found.

Endorepellin blocks the angiogenic activity of VEGF (FIG. 2d), as determined by the chicken chorioallantoic membrane (CAM) assay. Thus, HUVEC migration is inhibited, with the subsequent decrease in angiogenesis in vivo. In the presence of VEGF, the characteristic spoke wheel-like vessel formation is induced towards the sponge. In the presence of endorepellin, the vessel sprouts are markedly reduced to a level comparable to the negative control. These experiments are repeated several times with identical results.

To further investigate the role of endorepellin in angiogenesis, HUVEC tube formation is utilized in a collagen matrix (33). The results show a capillary-like network formation in the control HUVECs (FIG. 3a), which is visible at 4 h and remained constant for up to 24 h. In contrast, endorepellin causes a complete block of tube-like formation (FIG. 3b), whereas no significant effects were obtained with endostatin (FIG. 3c). Surprisingly, endorepellin activity is counteracted by endostatin (FIG. 3d). It should be noted that the concentration of endorepellin used in these assays (μg/ml) is even lower than that used for the migration assays. In fact, while in the latter, full inhibition is detected at 300-500 ng endorepellin/10⁴ HUVEC (30-50 pg/cell), in the former, full inhibition is achieved at 10 μg endorepellin/4×10⁵ HUVEC (25 pg/cell). Thus, the present invention reveals that endorepellin is a powerful blocker of angiogenesis in three independent assays commonly used to investigate angiogenesis.

Biological Effects of Endostatin/Endorepellin Interaction

To further investigate the interaction between endostatin and endorepellin, several VEGF-induced HUVEC migration experiments are performed in which the amount of endorepellin is kept constant while the amount of endostatin is proportionally increased. Two concentrations of endorepellin are used, 1.2 and 3.7 nM (100 and 300 ng/ml, respectively), that give suboptimal and optimal inhibition of HUVEC migration. When endostatin and endorepellin are concurrently present, there is a significant and dose-dependent inhibition of their activity (FIGS. 4a and b). By plotting the percentage of migrated cells, derived from normalized data of five independent experiments against the increasing molar ratios of endostatin/endorepellin, it is evident that maximal inhibition is achieved at about 1:1 molar ratio; the inhibition subsequently declined (not shown). Thus, the interaction between endostatin and endorepellin leads to a marked attenuation of their anti-angiogenic activity.

Endorepellin has Counter-adhesive Properties for Endothelial Cells

A number of bioactive fragments of extracellular matrix proteins exhibit counter-adhesive activities, that is, they disrupt cell-matrix interactions (34). It has been previously shown that domain V of perlecan is adhesive for several cell lines vis á vis fibronectin, but not for others (35). To address this point, endorepellin is tested for its ability to mediate HUVEC adhesion. The result is a complete lack of HUVEC adhesion to either endorepellin or BSA, in contrast to a robust adhesion to fibronectin (FIG. 5a) or collagen type I. In competition experiments, in which HUVECs are challenged with increasing amounts of endorepellin, a progressive inhibition of HUVEC attachment occurs. Within minutes, the cells rounded up and began to detach in a dose-dependent manner (FIG. 5b). Several experiments on fibrillar collagen or plastic are performed and endorepellin consistantly prevented HUVEC binding to either substratum, with IC₅₀ values of 5-20 nM. In contrast, endostatin does not show any interference with endothelial cell attachment to either fibronectin or collagen I.

To verify that this anti-adhesive property of endorepellin is not limited to endothelial cells, HT1080 fibrosarcoma cells, which do not bind to murine domain V (35), and WiDr colon carcinoma cells (14) are tested. Endorepellin does not support adhesion of either HT1080 fibrosarcoma cells or WiDr colon carcinoma cells (FIGS. 5c and d). Moreover, the specificity of endorepellin counter-adhesive properties is demonstrated by the efficient displacement of HT1080 and WiDr attachment to fibronectin using increasing concentrations of recombinant endorepellin (IC₅₀ of 110 and 40 nM, respectively; FIG 5 e). In contrast, endostatin does not significantly affect the adhesion of either cell line (FIG. 5f).

Adhesion assays also are performed using fibronectin and two concentrations of collagen type I with A431 squamous carcinoma cells, which were previously shown to adhere to murine domain V (58% of the values with a mean attachment value of 52±4% (Table 1) were obtained). Mouse M2 melanoma and human MCF7 breast carcinoma cells are also analyzed for adhesion in the presence of endorepellin. The resultant attachment values are <5% for mouse M2 melanoma cells and 50±7% for human MCF7 breast carcinoma cells (Table 1). Thus, endorepellin is a powerful anti-adhesive factor for endothelial cells and certain tumor cells.

Discussion

The present invention discloses screening, in vivo, for molecules that interact with the perlecan protein core. It is discovered that the cDNA encoding endostatin, the anti-angiogenic C-terminal fragment derived from collagen type XVIII (28), interacts strongly with the perlecan protein core. It previsously has been shown, using an in vitro cell-free system, that the entire perlecan binds to endostatin, presumably via the heparan sulfate chains (36,37). These results are confirmed herein. In addition, the present invention shows that a distinct subdomain of perlecan protein core binds specifically to endostatin.

Using a battery of deletion mutants, the precise binding site of endostatin to perlecan is mapped to the second laminin-like G (LG) domain of perlecan domain V. Since perlecan and type XVIII collagen/endostatin co-distribute in basement membranes (29, 30, 36) and endostatin binds in situ to vascular basement membranes independently of heparan sulfate (38), domain V is a likely binding site for endostatin in vivo.

Using HUVEC migration assays the present invention reveals that the interaction between endostatin and domain V counteracts the anti-angiogenic effects of endostatin. Since perlecan domain V itself is a powerful anti-angiogenic factor it is named herein “endorepellin”. Endorepellin is active at nanomolar concentrations and is a potent inhibitor of angiogenesis in three independent assays commonly used to study angiogenesis: endothelial cell migration through fibrillar collagen, collagen-induced capillary-like formation, and growth of blood vessels in the chorioallantoic membrane. Interestingly, the action of endorepellin is as strong as endostatin in inhibiting HUVEC migration. Endorepellin interferes with the adhesive properties of endothelial cells for various substrata, including, but not limited to, fibronectin and fibrillar collagen, and also is anti-adhesive for certain tumor cells derived from colon, neuroectoderm or mesenchyme. This is in agreement with previous studies showing anti-adhesive properties for perlecan in hematopoietic (24), mesangial (25) and smooth muscle (39) cells, and a role for perlecan in the suppression of growth and invasion in fibrosarcoma cells (40). However, while endorepellin inhibits tube formation and prevents binding to fibronectin and other substrata, endostatin does not.

Powerful angiogenesis inhibitors are proteolytically-processed forms of basement membrane collagens types IV, XV and XVIII, the latter two being chondroitin and HSPGs, respectively (41). Moreover, proteolytic remodeling of the extracellular matrix can expose cryptic sites within collagen type IV that are required for angiogenesis in vivo (42). Thus, it is likely that perlecan undergoes a similar proteolytic processing in vivo, thereby liberating endorepellin.

The modular nature of the perlecan protein core is particularly well suited for selective proteolysis (12, 41, 43) and subsequent release of peptides with biological activity. There are two lines of evidence that support this scenario. First, in 293-EBNA cells a natural proteolytic cleavage of endorepellin of ˜25 kDa is detected which binds to the Ni-NTA column and is also reactive with the anti-His6 antibody, indicating that it represents LG3. A similar size band previously was shown to represent a proteolytic fragment of murine domain V generated by cleavage just before the beginning of LG3 (9,35). This protease-sensitive region, which starts with the sequence DAPGQY (SEQ. ID. NO: 1), is completely conserved between mouse (3) and human (4), thus demonstrating that a specific cleavage of Asn-Asp bond (at position 3514/3515 and 4196/4197, for the mouse and human counterpart, respectively) had occurred near the N-terminus of LG3. Mutational analysis indicated that Asp but not Asn is crucial for processing of mouse endorepellin (44), possibly by a specific, yet to be discovered, Asp-N endoproteinase.

The second line of evidence is that an identical proteolytic fragment of ˜25 kDa, cleaved at the same position as the mouse, was detected in the urine of patients with end-stage renal failure (45). This indicates that the LG3 module is present in human serum at relatively high concentrations since this LG3 was found at concentrations of ˜10 mg/L of urine (45). Circulating forms of endorepellin may be involved in the homeostatic control of angiogenesis as proposed previously for endostatin, whose levels can reach 0.3 mg/L of blood (36).

Recent experimental tests on tumor-bearing animals are encouraging because protein-based inhibitors, such as endostatin, have three major advantages: 1) they can reduce the tumors to a bearable size, 2) they do not induce resistance, and 3) their toxicity is low (41). Endorepellin is a novel, natural inhibitor of angiogeneis and its use in cancer therapy has additional advantages insofar as endorepellin also exerts an anti-adhesive action on certain tumor cells. The invention disclosed herein presents a method wherein endorepellin inhibits angiogenesis and is used in the treatment of angiogenesis-mediated diseases (supra), such as cancer. Endorepellin also is used in conjunction with standard therapeutic agents to treat angiogenesis-dependent diseases, thereby enhancing the efficacy of current methodologies.

The foregoing descriptions of specific embodiments of the present invention have been presented for the purposes of illustration and description. They are not intended to be exhaustive or to limit the invention to the precise forms disclosed, and obviously many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention and its practical application to thereby enable others skilled in the art to best utilize the invention and various embodiments with various modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

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10 1 6 PRT human frag. 3927-4181 1 Asp Ala Pro Gly Gln Tyr 1 5 2 4391 PRT human 2 Met Gly Trp Arg Ala Pro Gly Ala Leu Leu Leu Ala Leu Leu Leu His 1 5 10 15 Gly Arg Leu Leu Ala Val Thr His Gly Leu Arg Ala Tyr Asp Gly Leu 20 25 30 Ser Leu Pro Glu Asp Ile Glu Thr Val Thr Ala Ser Gln Met Arg Trp 35 40 45 Thr His Ser Tyr Leu Ser Asp Asp Glu Tyr Met Leu Ala Asp Ser Ile 50 55 60 Ser Gly Asp Asp Leu Gly Ser Gly Asp Leu Gly Ser Gly Asp Phe Gln 65 70 75 80 Met Val Tyr Phe Arg Ala Leu Val Asn Phe Thr Arg Ser Ile Glu Tyr 85 90 95 Ser Pro Gln Leu Glu Asp Ala Gly Ser Arg Glu Phe Arg Glu Val Ser 100 105 110 Glu Ala Val Val Asp Thr Leu Glu Ser Glu Tyr Leu Lys Ile Pro Gly 115 120 125 Asp Gln Val Val Ser Val Val Phe Ile Lys Glu Leu Asp Gly Trp Val 130 135 140 Phe Val Glu Leu Asp Val Gly Ser Glu Gly Asn Ala Asp Gly Ala Gln 145 150 155 160 Ile Gln Glu Met Leu Leu Arg Val Ile Ser Ser Gly Ser Val Ala Ser 165 170 175 Tyr Val Thr Ser Pro Gln Gly Phe Gln Phe Arg Arg Leu Gly Thr Val 180 185 190 Pro Gln Phe Pro Arg Ala Cys Thr Glu Ala Glu Phe Ala Cys His Ser 195 200 205 Tyr Asn Glu Cys Val Ala Leu Glu Tyr Arg Cys Asp Arg Arg Pro Asp 210 215 220 Cys Arg Asp Met Ser Asp Glu Leu Asn Cys Glu Glu Pro Val Leu Gly 225 230 235 240 Ile Ser Pro Thr Phe Ser Leu Leu Val Glu Thr Thr Ser Leu Pro Pro 245 250 255 Arg Pro Glu Thr Thr Ile Met Arg Gln Pro Pro Val Thr His Ala Pro 260 265 270 Gln Pro Leu Leu Pro Gly Ser Val Arg Pro Leu Pro Cys Gly Pro Gln 275 280 285 Glu Ala Ala Cys Arg Asn Gly His Cys Ile Pro Arg Asp Tyr Leu Cys 290 295 300 Asp Gly Gln Glu Asp Cys Glu Asp Gly Ser Asp Glu Leu Asp Cys Gly 305 310 315 320 Pro Pro Pro Pro Cys Glu Pro Asn Glu Phe Pro Cys Gly Asn Gly His 325 330 335 Cys Ala Leu Lys Leu Trp Arg Cys Asp Gly Asp Phe Asp Cys Glu Asp 340 345 350 Arg Thr Asp Glu Ala Asn Cys Pro Thr Lys Arg Pro Glu Glu Val Cys 355 360 365 Gly Pro Thr Gln Phe Arg Cys Val Ser Thr Asn Met Cys Ile Pro Ala 370 375 380 Ser Phe His Cys Asp Glu Glu Ser Asp Cys Pro Asp Arg Ser Asp Glu 385 390 395 400 Phe Gly Cys Met Pro Pro Gln Val Val Thr Pro Pro Arg Glu Ser Ile 405 410 415 Gln Ala Ser Arg Gly Gln Thr Val Thr Phe Thr Cys Val Ala Ile Gly 420 425 430 Val Pro Thr Pro Ile Ile Asn Trp Arg Leu Asn Trp Gly His Ile Pro 435 440 445 Ser His Pro Arg Val Thr Val Thr Ser Glu Gly Gly Arg Gly Thr Leu 450 455 460 Ile Ile Arg Asp Val Lys Glu Ser Asp Gln Gly Ala Tyr Thr Cys Glu 465 470 475 480 Ala Met Asn Ala Arg Gly Met Val Phe Gly Ile Pro Asp Gly Val Leu 485 490 495 Glu Leu Val Pro Gln Arg Gly Pro Cys Pro Asp Gly His Phe Tyr Leu 500 505 510 Glu His Ser Ala Ala Cys Leu Pro Cys Phe Cys Phe Gly Ile Thr Ser 515 520 525 Val Cys Gln Ser Thr Arg Arg Phe Arg Asp Gln Ile Arg Leu Arg Phe 530 535 540 Asp Gln Pro Asp Asp Phe Lys Gly Val Asn Val Thr Met Pro Ala Gln 545 550 555 560 Pro Gly Thr Pro Pro Leu Ser Ser Thr Gln Leu Gln Ile Asp Pro Ser 565 570 575 Leu His Glu Phe Gln Leu Val Asp Leu Ser Arg Arg Phe Leu Val His 580 585 590 Asp Ser Phe Trp Ala Leu Pro Glu Gln Phe Leu Gly Asn Lys Val Asp 595 600 605 Ser Tyr Gly Gly Ser Leu Arg Tyr Asn Val Arg Tyr Glu Leu Ala Arg 610 615 620 Gly Met Leu Glu Pro Val Gln Arg Pro Asp Val Val Leu Val Gly Ala 625 630 635 640 Gly Tyr Arg Leu Leu Ser Arg Gly His Thr Pro Thr Gln Pro Gly Ala 645 650 655 Leu Asn Gln Arg Gln Val Gln Phe Ser Glu Glu His Trp Val His Glu 660 665 670 Ser Gly Arg Pro Val Gln Arg Ala Glu Leu Leu Gln Val Leu Gln Ser 675 680 685 Leu Glu Ala Val Leu Ile Gln Thr Val Tyr Asn Thr Lys Met Ala Ser 690 695 700 Val Gly Leu Ser Asp Ile Ala Met Asp Thr Thr Val Thr His Ala Thr 705 710 715 720 Ser His Gly Arg Ala His Ser Val Glu Glu Cys Arg Cys Pro Ile Gly 725 730 735 Tyr Ser Gly Leu Ser Cys Glu Ser Cys Asp Ala His Phe Thr Arg Val 740 745 750 Pro Gly Gly Pro Tyr Leu Gly Thr Cys Ser Gly Cys Ser Cys Asn Gly 755 760 765 His Ala Ser Ser Cys Asp Pro Val Tyr Gly His Cys Leu Asn Cys Gln 770 775 780 His Asn Thr Glu Gly Pro Gln Cys Asn Lys Cys Lys Ala Gly Phe Phe 785 790 795 800 Gly Asp Ala Met Lys Ala Thr Ala Thr Ser Cys Arg Pro Cys Pro Cys 805 810 815 Pro Tyr Ile Asp Ala Ser Arg Arg Phe Ser Asp Thr Cys Phe Leu Asp 820 825 830 Thr Asp Gly Gln Ala Thr Cys Asp Ala Cys Ala Pro Gly Tyr Thr Gly 835 840 845 Arg Arg Cys Glu Ser Cys Ala Pro Gly Tyr Glu Gly Asn Pro Ile Gln 850 855 860 Pro Gly Gly Lys Cys Arg Pro Val Asn Gln Glu Ile Val Arg Cys Asp 865 870 875 880 Glu Arg Gly Ser Met Gly Thr Ser Gly Glu Ala Cys Arg Cys Lys Asn 885 890 895 Asn Val Val Gly Arg Leu Cys Asn Glu Cys Ala Asp Gly Ser Phe His 900 905 910 Leu Ser Thr Arg Asn Pro Asp Gly Cys Leu Lys Cys Phe Cys Met Gly 915 920 925 Val Ser Arg His Cys Thr Ser Ser Ser Trp Ser Arg Ala Gln Leu His 930 935 940 Gly Ala Ser Glu Glu Pro Gly His Phe Ser Leu Thr Asn Ala Ala Ser 945 950 955 960 Thr His Thr Thr Asn Glu Gly Ile Phe Ser Pro Thr Pro Gly Glu Leu 965 970 975 Gly Phe Ser Ser Phe His Arg Leu Leu Ser Gly Pro Tyr Phe Trp Ser 980 985 990 Leu Pro Ser Arg Phe Leu Gly Asp Lys Val Thr Ser Tyr Gly Gly Glu 995 1000 1005 Leu Arg Phe Thr Val Thr Gln Arg Ser Gln Pro Gly Ser Thr Pro Leu 1010 1015 1020 His Gly Gln Pro Leu Val Val Leu Gln Gly Asn Asn Ile Ile Leu Glu 1025 1030 1035 1040 His His Val Ala Gln Glu Pro Ser Pro Gly Gln Pro Ser Thr Phe Ile 1045 1050 1055 Val Pro Phe Arg Glu Gln Ala Trp Gln Arg Pro Asp Gly Gln Pro Ala 1060 1065 1070 Thr Arg Glu His Leu Leu Met Ala Leu Ala Gly Ile Asp Thr Leu Leu 1075 1080 1085 Ile Arg Ala Ser Tyr Ala Gln Gln Pro Ala Glu Ser Arg Val Ser Gly 1090 1095 1100 Ile Ser Met Asp Val Ala Val Pro Glu Glu Thr Gly Gln Asp Pro Ala 1105 1110 1115 1120 Leu Glu Val Glu Gln Cys Ser Cys Pro Pro Gly Tyr Arg Gly Pro Ser 1125 1130 1135 Cys Gln Asp Cys Asp Thr Gly Tyr Thr Arg Thr Pro Ser Gly Leu Tyr 1140 1145 1150 Leu Gly Thr Cys Glu Arg Cys Ser Cys His Gly His Ser Glu Ala Cys 1155 1160 1165 Glu Pro Glu Thr Gly Ala Cys Gln Gly Cys Gln His His Thr Glu Gly 1170 1175 1180 Pro Arg Cys Glu Gln Cys Gln Pro Gly Tyr Tyr Gly Asp Ala Gln Arg 1185 1190 1195 1200 Gly Thr Pro Gln Asp Cys Gln Leu Cys Pro Cys Tyr Gly Asp Pro Ala 1205 1210 1215 Ala Gly Gln Ala Ala His Thr Cys Phe Leu Asp Thr Asp Gly His Pro 1220 1225 1230 Thr Cys Asp Ala Cys Ser Pro Gly His Ser Gly Arg His Cys Glu Arg 1235 1240 1245 Cys Ala Pro Gly Tyr Tyr Gly Asn Pro Ser Gln Gly Gln Pro Cys Gln 1250 1255 1260 Arg Asp Ser Gln Val Pro Gly Pro Ile Gly Cys Asn Cys Asp Pro Gln 1265 1270 1275 1280 Gly Ser Val Ser Ser Gln Cys Asp Ala Ala Gly Gln Cys Gln Cys Lys 1285 1290 1295 Ala Gln Val Glu Gly Leu Thr Cys Ser His Cys Arg Pro His His Phe 1300 1305 1310 His Leu Ser Ala Ser Asn Pro Asp Gly Cys Leu Pro Cys Phe Cys Met 1315 1320 1325 Gly Ile Thr Gln Gln Cys Ala Ser Ser Ala Tyr Thr Arg His Leu Ile 1330 1335 1340 Ser Thr His Phe Ala Pro Gly Asp Phe Gln Gly Phe Ala Leu Val Asn 1345 1350 1355 1360 Pro Gln Arg Asn Ser Arg Leu Thr Gly Glu Phe Thr Val Glu Pro Val 1365 1370 1375 Pro Glu Gly Ala Gln Leu Ser Phe Gly Asn Phe Ala Gln Leu Gly His 1380 1385 1390 Glu Ser Phe Tyr Trp Gln Leu Pro Glu Thr Tyr Gln Gly Asp Lys Val 1395 1400 1405 Ala Ala Tyr Gly Gly Lys Leu Arg Tyr Thr Leu Ser Tyr Thr Ala Gly 1410 1415 1420 Pro Gln Gly Ser Pro Leu Ser Asp Pro Asp Val Gln Ile Thr Gly Asn 1425 1430 1435 1440 Asn Ile Met Leu Val Ala Ser Gln Pro Ala Leu Gln Gly Pro Glu Arg 1445 1450 1455 Arg Ser Tyr Glu Ile Met Phe Arg Glu Glu Phe Trp Arg Arg Pro Asp 1460 1465 1470 Gly Gln Pro Ala Thr Arg Glu His Leu Leu Met Ala Leu Ala Asp Leu 1475 1480 1485 Asp Glu Leu Leu Ile Arg Ala Thr Phe Ser Ser Val Pro Leu Val Ala 1490 1495 1500 Ser Ile Ser Ala Val Ser Leu Glu Val Ala Gln Pro Gly Pro Ser Asn 1505 1510 1515 1520 Arg Pro Arg Ala Leu Glu Val Glu Glu Cys Arg Cys Pro Pro Gly Tyr 1525 1530 1535 Ile Gly Leu Ser Cys Gln Asp Cys Ala Pro Gly Tyr Thr Arg Thr Gly 1540 1545 1550 Ser Gly Leu Tyr Leu Gly His Cys Glu Leu Cys Glu Cys Asn Gly His 1555 1560 1565 Ser Asp Leu Cys His Pro Glu Thr Gly Ala Cys Ser Gln Cys Gln His 1570 1575 1580 Asn Ala Ala Gly Glu Phe Cys Glu Leu Cys Ala Pro Gly Tyr Tyr Gly 1585 1590 1595 1600 Asp Ala Thr Ala Gly Thr Pro Glu Asp Cys Gln Pro Cys Ala Cys Pro 1605 1610 1615 Leu Thr Asn Pro Glu Asn Met Phe Ser Arg Thr Cys Glu Ser Leu Gly 1620 1625 1630 Ala Gly Gly Tyr Arg Cys Thr Ala Cys Glu Pro Gly Tyr Thr Gly Gln 1635 1640 1645 Tyr Cys Glu Gln Cys Gly Pro Gly Tyr Val Gly Asn Pro Ser Val Gln 1650 1655 1660 Gly Gly Gln Cys Leu Pro Glu Thr Asn Gln Ala Pro Leu Val Val Glu 1665 1670 1675 1680 Val His Pro Ala Arg Ser Ile Val Pro Gln Gly Gly Ser His Ser Leu 1685 1690 1695 Arg Cys Gln Val Ser Gly Ser Pro Pro His Tyr Phe Tyr Trp Ser Arg 1700 1705 1710 Glu Asp Gly Arg Pro Val Pro Ser Gly Thr Gln Gln Arg His Gln Gly 1715 1720 1725 Ser Glu Leu His Phe Pro Ser Val Gln Pro Ser Asp Ala Gly Val Tyr 1730 1735 1740 Ile Cys Thr Cys Arg Asn Leu His Gln Ser Asn Thr Ser Arg Ala Glu 1745 1750 1755 1760 Leu Leu Val Thr Glu Ala Pro Ser Lys Pro Ile Thr Val Thr Val Glu 1765 1770 1775 Glu Gln Arg Ser Gln Ser Val Arg Pro Gly Ala Asp Val Thr Phe Ile 1780 1785 1790 Cys Thr Ala Lys Ser Lys Ser Pro Ala Tyr Thr Leu Val Trp Thr Arg 1795 1800 1805 Leu His Asn Gly Lys Leu Pro Thr Arg Ala Met Asp Phe Asn Gly Ile 1810 1815 1820 Leu Thr Ile Arg Asn Val Gln Leu Ser Asp Ala Gly Thr Tyr Val Cys 1825 1830 1835 1840 Thr Gly Ser Asn Met Phe Ala Met Asp Gln Gly Thr Ala Thr Leu His 1845 1850 1855 Val Gln Ala Ser Gly Thr Leu Ser Ala Pro Val Val Ser Ile His Pro 1860 1865 1870 Pro Gln Leu Thr Val Gln Pro Gly Gln Leu Ala Glu Phe Arg Cys Ser 1875 1880 1885 Ala Thr Gly Ser Pro Thr Pro Thr Leu Glu Trp Thr Gly Gly Pro Gly 1890 1895 1900 Gly Gln Leu Pro Ala Lys Ala Gln Ile His Gly Gly Ile Leu Arg Leu 1905 1910 1915 1920 Pro Ala Val Glu Pro Thr Asp Gln Ala Gln Tyr Leu Cys Arg Ala His 1925 1930 1935 Ser Ser Ala Gly Gln Gln Val Ala Arg Ala Val Leu His Val His Gly 1940 1945 1950 Gly Gly Gly Pro Arg Val Gln Val Ser Pro Glu Arg Thr Gln Val His 1955 1960 1965 Ala Gly Arg Thr Val Arg Leu Tyr Cys Arg Ala Ala Gly Val Pro Ser 1970 1975 1980 Ala Thr Ile Thr Trp Arg Lys Glu Gly Gly Ser Leu Pro Pro Gln Ala 1985 1990 1995 2000 Arg Ser Glu Arg Thr Asp Ile Ala Thr Leu Leu Ile Pro Ala Ile Thr 2005 2010 2015 Thr Ala Asp Ala Gly Phe Tyr Leu Cys Val Ala Thr Ser Pro Ala Gly 2020 2025 2030 Thr Ala Gln Ala Arg Met Gln Val Val Val Leu Ser Ala Ser Asp Ala 2035 2040 2045 Ser Pro Pro Gly Val Lys Ile Glu Ser Ser Ser Pro Ser Val Thr Glu 2050 2055 2060 Gly Gln Thr Leu Asp Leu Asn Cys Val Val Ala Gly Ser Ala His Ala 2065 2070 2075 2080 Gln Val Thr Trp Tyr Arg Arg Gly Gly Ser Leu Pro Pro His Thr Gln 2085 2090 2095 Val His Gly Ser Arg Leu Arg Leu Pro Gln Val Ser Pro Ala Asp Ser 2100 2105 2110 Gly Glu Tyr Val Cys Arg Val Glu Asn Gly Ser Gly Pro Lys Glu Ala 2115 2120 2125 Ser Ile Thr Val Ser Val Leu His Gly Thr His Ser Gly Pro Ser Tyr 2130 2135 2140 Thr Pro Val Pro Gly Ser Thr Arg Pro Ile Arg Ile Glu Pro Ser Ser 2145 2150 2155 2160 Ser His Val Ala Glu Gly Gln Thr Leu Asp Leu Asn Cys Val Val Pro 2165 2170 2175 Gly Gln Ala His Ala Gln Val Thr Trp His Lys Arg Gly Gly Ser Leu 2180 2185 2190 Pro Ala Arg His Gln Thr His Gly Ser Leu Leu Arg Leu His Gln Val 2195 2200 2205 Thr Pro Ala Asp Ser Gly Glu Tyr Val Cys His Val Val Gly Thr Ser 2210 2215 2220 Gly Pro Leu Glu Ala Ser Val Leu Val Thr Ile Glu Ala Ser Val Ile 2225 2230 2235 2240 Pro Gly Pro Ile Pro Pro Val Arg Ile Glu Ser Ser Ser Ser Thr Val 2245 2250 2255 Ala Glu Gly Gln Thr Leu Asp Leu Ser Cys Val Val Ala Gly Gln Ala 2260 2265 2270 His Ala Gln Val Thr Trp Tyr Lys Arg Gly Gly Ser Leu Pro Ala Arg 2275 2280 2285 His Gln Val Arg Gly Ser Arg Leu Tyr Ile Phe Gln Ala Ser Pro Ala 2290 2295 2300 Asp Ala Gly Gln Tyr Val Cys Arg Ala Ser Asn Gly Met Glu Ala Ser 2305 2310 2315 2320 Ile Thr Val Thr Val Thr Gly Thr Gln Gly Ala Asn Leu Ala Tyr Pro 2325 2330 2335 Ala Gly Ser Thr Gln Pro Ile Arg Ile Glu Pro Ser Ser Ser Gln Val 2340 2345 2350 Ala Glu Gly Gln Thr Leu Asp Leu Asn Cys Val Val Pro Gly Gln Ser 2355 2360 2365 His Ala Gln Val Thr Trp His Lys Arg Gly Gly Ser Leu Pro Val Arg 2370 2375 2380 His Gln Thr His Gly Ser Leu Leu Arg Leu Tyr Gln Ala Ser Pro Ala 2385 2390 2395 2400 Asp Ser Gly Glu Tyr Val Cys Arg Val Leu Gly Ser Ser Val Pro Leu 2405 2410 2415 Glu Ala Ser Val Leu Val Thr Ile Glu Pro Ala Gly Ser Val Pro Ala 2420 2425 2430 Leu Gly Val Thr Pro Thr Val Arg Ile Glu Ser Ser Ser Ser Gln Val 2435 2440 2445 Ala Glu Gly Gln Thr Leu Asp Leu Asn Cys Leu Val Ala Gly Gln Ala 2450 2455 2460 His Ala Gln Val Thr Trp His Lys Arg Gly Gly Ser Leu Pro Ala Arg 2465 2470 2475 2480 His Gln Val His Gly Ser Arg Leu Arg Leu Leu Gln Val Thr Pro Ala 2485 2490 2495 Asp Ser Gly Glu Tyr Val Cys Arg Val Val Gly Ser Ser Gly Thr Gln 2500 2505 2510 Glu Ala Ser Val Leu Val Thr Ile Gln Gln Arg Leu Ser Gly Ser His 2515 2520 2525 Ser Gln Gly Val Ala Tyr Pro Val Arg Ile Glu Ser Ser Ser Ala Ser 2530 2535 2540 Leu Ala Asn Gly His Thr Leu Asp Leu Asn Cys Leu Val Ala Ser Gln 2545 2550 2555 2560 Ala Pro His Thr Ile Thr Trp Tyr Lys Arg Gly Gly Ser Leu Pro Ser 2565 2570 2575 Arg His Gln Ile Val Gly Ser Arg Leu Arg Ile Pro Gln Val Thr Pro 2580 2585 2590 Ala Asp Ser Gly Glu Tyr Val Cys His Val Ser Asn Gly Ala Gly Ser 2595 2600 2605 Arg Glu Thr Ser Leu Ile Val Thr Ile Gln Gly Ser Gly Ser Ser His 2610 2615 2620 Val Pro Ser Val Ser Pro Pro Ile Arg Ile Glu Ser Ser Ser Pro Thr 2625 2630 2635 2640 Val Val Glu Gly Gln Thr Leu Asp Leu Asn Cys Val Val Ala Arg Gln 2645 2650 2655 Pro Gln Ala Ile Ile Thr Trp Tyr Lys Arg Gly Gly Ser Leu Pro Ser 2660 2665 2670 Arg His Gln Thr His Gly Ser His Leu Arg Leu His Gln Met Ser Val 2675 2680 2685 Ala Asp Ser Gly Glu Tyr Val Cys Arg Ala Asn Asn Asn Ile Asp Ala 2690 2695 2700 Leu Glu Ala Ser Ile Val Ile Ser Val Ser Pro Ser Ala Gly Ser Pro 2705 2710 2715 2720 Ser Ala Pro Gly Ser Ser Met Pro Ile Arg Ile Glu Ser Ser Ser Ser 2725 2730 2735 His Val Ala Glu Gly Glu Thr Leu Asp Leu Asn Cys Val Val Pro Gly 2740 2745 2750 Gln Ala His Ala Gln Val Thr Trp His Lys Arg Gly Gly Ser Leu Pro 2755 2760 2765 Ser His His Gln Thr Arg Gly Ser Arg Leu Arg Leu His His Val Ser 2770 2775 2780 Pro Ala Asp Ser Gly Glu Tyr Val Cys Arg Val Met Gly Ser Ser Gly 2785 2790 2795 2800 Pro Leu Glu Ala Ser Val Leu Val Thr Ile Glu Ala Ser Gly Ser Ser 2805 2810 2815 Ala Val His Val Pro Ala Pro Gly Gly Ala Pro Pro Ile Arg Ile Glu 2820 2825 2830 Pro Ser Ser Ser Arg Val Ala Glu Gly Gln Thr Leu Asp Leu Lys Cys 2835 2840 2845 Val Val Pro Gly Gln Ala His Ala Gln Val Thr Trp His Lys Arg Gly 2850 2855 2860 Gly Asn Leu Pro Ala Arg His Gln Val His Gly Pro Leu Leu Arg Leu 2865 2870 2875 2880 Asn Gln Val Ser Pro Ala Asp Ser Gly Glu Tyr Ser Cys Gln Val Thr 2885 2890 2895 Gly Ser Ser Gly Thr Leu Glu Ala Ser Val Leu Val Thr Ile Glu Pro 2900 2905 2910 Ser Ser Pro Gly Pro Ile Pro Ala Pro Gly Leu Ala Gln Pro Ile Tyr 2915 2920 2925 Ile Glu Ala Ser Ser Ser His Val Thr Glu Gly Gln Thr Leu Asp Leu 2930 2935 2940 Asn Cys Val Val Pro Gly Gln Ala His Ala Gln Val Thr Trp Tyr Lys 2945 2950 2955 2960 Arg Gly Gly Ser Leu Pro Ala Arg His Gln Thr His Gly Ser Gln Leu 2965 2970 2975 Arg Leu His Leu Val Ser Pro Ala Asp Ser Gly Glu Tyr Val Cys Arg 2980 2985 2990 Ala Ala Ser Gly Pro Gly Pro Glu Gln Glu Ala Ser Phe Thr Val Thr 2995 3000 3005 Val Pro Pro Ser Glu Gly Ser Ser Tyr Arg Leu Arg Ser Pro Val Ile 3010 3015 3020 Ser Ile Asp Pro Pro Ser Ser Thr Val Gln Gln Gly Gln Asp Ala Ser 3025 3030 3035 3040 Phe Lys Cys Leu Ile His Asp Gly Ala Ala Pro Ile Ser Leu Glu Trp 3045 3050 3055 Lys Thr Arg Asn Gln Glu Leu Glu Asp Asn Val His Ile Ser Pro Asn 3060 3065 3070 Gly Ser Ile Ile Thr Ile Val Gly Thr Arg Pro Ser Asn His Gly Thr 3075 3080 3085 Tyr Arg Cys Val Ala Ser Asn Ala Tyr Gly Val Ala Gln Ser Val Val 3090 3095 3100 Asn Leu Ser Val His Gly Pro Pro Thr Val Ser Val Leu Pro Glu Gly 3105 3110 3115 3120 Pro Val Trp Val Lys Val Gly Lys Ala Val Thr Leu Glu Cys Val Ser 3125 3130 3135 Ala Gly Glu Pro Arg Ser Ser Ala Arg Trp Thr Arg Ile Ser Ser Thr 3140 3145 3150 Pro Ala Lys Leu Glu Gln Arg Thr Tyr Gly Leu Met Asp Ser His Ala 3155 3160 3165 Val Leu Gln Ile Ser Ser Ala Lys Pro Ser Asp Ala Gly Thr Tyr Val 3170 3175 3180 Cys Leu Ala Gln Asn Ala Leu Gly Thr Ala Gln Lys Gln Val Glu Val 3185 3190 3195 3200 Ile Val Asp Thr Gly Ala Met Ala Pro Gly Ala Pro Gln Val Gln Ala 3205 3210 3215 Glu Glu Ala Glu Leu Thr Val Glu Ala Gly His Thr Ala Thr Leu Arg 3220 3225 3230 Cys Ser Ala Thr Gly Ser Pro Ala Pro Thr Ile His Trp Ser Lys Leu 3235 3240 3245 Arg Ser Pro Leu Pro Trp Gln His Arg Leu Glu Gly Asp Thr Leu Ile 3250 3255 3260 Ile Pro Arg Val Ala Gln Gln Asp Ser Gly Gln Tyr Ile Cys Asn Ala 3265 3270 3275 3280 Thr Ser Pro Ala Gly His Ala Glu Ala Thr Ile Ile Leu His Val Glu 3285 3290 3295 Ser Pro Pro Tyr Ala Thr Thr Val Pro Glu His Ala Ser Val Gln Ala 3300 3305 3310 Gly Glu Thr Val Gln Leu Gln Cys Leu Ala His Gly Thr Pro Pro Leu 3315 3320 3325 Thr Phe Gln Trp Ser Arg Val Gly Ser Ser Leu Pro Gly Arg Ala Thr 3330 3335 3340 Ala Arg Asn Glu Leu Leu His Phe Glu Arg Ala Ala Pro Glu Asp Ser 3345 3350 3355 3360 Gly Arg Tyr Arg Cys Arg Val Thr Asn Lys Val Gly Ser Ala Glu Ala 3365 3370 3375 Phe Ala Gln Leu Leu Val Gln Gly Pro Pro Gly Ser Leu Pro Ala Thr 3380 3385 3390 Ser Ile Pro Ala Gly Ser Thr Pro Thr Val Gln Val Thr Pro Gln Leu 3395 3400 3405 Glu Thr Lys Ser Ile Gly Ala Ser Val Glu Phe His Cys Ala Val Pro 3410 3415 3420 Ser Asp Gln Gly Thr Gln Leu Arg Trp Phe Lys Glu Gly Gly Gln Leu 3425 3430 3435 3440 Pro Pro Gly His Ser Val Gln Asp Gly Val Leu Arg Ile Gln Asn Leu 3445 3450 3455 Asp Gln Ser Cys Gln Gly Thr Tyr Ile Cys Gln Ala His Gly Pro Trp 3460 3465 3470 Gly Lys Ala Gln Ala Ser Ala Gln Leu Val Ile Gln Ala Leu Pro Ser 3475 3480 3485 Val Leu Ile Asn Ile Arg Thr Ser Val Gln Thr Val Val Val Gly His 3490 3495 3500 Ala Val Glu Phe Glu Cys Leu Ala Leu Gly Asp Pro Lys Pro Gln Val 3505 3510 3515 3520 Thr Trp Ser Lys Val Gly Gly His Leu Arg Pro Gly Ile Val Gln Ser 3525 3530 3535 Gly Gly Val Val Arg Ile Ala His Val Glu Leu Ala Asp Ala Gly Gln 3540 3545 3550 Tyr Arg Cys Thr Ala Thr Asn Ala Ala Gly Thr Thr Gln Ser His Val 3555 3560 3565 Leu Leu Leu Val Gln Ala Leu Pro Gln Ile Ser Met Pro Gln Glu Val 3570 3575 3580 Arg Val Pro Ala Gly Ser Ala Ala Val Phe Pro Cys Ile Ala Ser Gly 3585 3590 3595 3600 Tyr Pro Thr Pro Asp Ile Ser Trp Ser Lys Leu Asp Gly Ser Leu Pro 3605 3610 3615 Pro Asp Ser Arg Leu Glu Asn Asn Met Leu Met Leu Pro Ser Val Arg 3620 3625 3630 Pro Gln Asp Ala Gly Thr Tyr Val Cys Thr Ala Thr Asn Arg Gln Gly 3635 3640 3645 Lys Val Lys Ala Phe Ala His Leu Gln Val Pro Glu Arg Val Val Pro 3650 3655 3660 Tyr Phe Thr Gln Thr Pro Tyr Ser Phe Leu Pro Leu Pro Thr Ile Lys 3665 3670 3675 3680 Asp Ala Tyr Arg Lys Phe Glu Ile Lys Ile Thr Phe Arg Pro Asp Ser 3685 3690 3695 Ala Asp Gly Met Leu Leu Tyr Asn Gly Gln Lys Arg Val Pro Gly Ser 3700 3705 3710 Pro Thr Asn Leu Ala Asn Arg Gln Pro Asp Phe Ile Ser Phe Gly Leu 3715 3720 3725 Val Gly Gly Arg Pro Glu Phe Arg Phe Asp Ala Gly Ser Gly Met Ala 3730 3735 3740 Thr Ile Arg His Pro Thr Pro Leu Ala Leu Gly His Phe His Thr Val 3745 3750 3755 3760 Thr Leu Leu Arg Ser Leu Thr Gln Gly Ser Leu Ile Val Gly Asp Leu 3765 3770 3775 Ala Pro Val Asn Gly Thr Ser Gln Gly Lys Phe Gln Gly Leu Asp Leu 3780 3785 3790 Asn Glu Glu Leu Tyr Leu Gly Gly Tyr Pro Asp Tyr Gly Ala Ile Pro 3795 3800 3805 Lys Ala Gly Leu Ser Ser Gly Phe Ile Gly Cys Val Arg Glu Leu Arg 3810 3815 3820 Ile Gln Gly Glu Glu Ile Val Phe His Asp Leu Asn Leu Thr Ala His 3825 3830 3835 3840 Gly Ile Ser His Cys Pro Thr Cys Arg Asp Arg Pro Cys Gln Asn Gly 3845 3850 3855 Gly Gln Cys His Asp Ser Glu Ser Ser Ser Tyr Val Cys Val Cys Pro 3860 3865 3870 Ala Gly Phe Thr Gly Ser Arg Cys Glu His Ser Gln Ala Leu His Cys 3875 3880 3885 His Pro Glu Ala Cys Gly Pro Asp Ala Thr Cys Val Asn Arg Pro Asp 3890 3895 3900 Gly Arg Gly Tyr Thr Cys Arg Cys His Leu Gly Arg Ser Gly Leu Arg 3905 3910 3915 3920 Cys Glu Glu Gly Val Thr Val Thr Thr Pro Ser Leu Ser Gly Ala Gly 3925 3930 3935 Ser Tyr Leu Ala Leu Pro Ala Leu Thr Asn Thr His His Glu Leu Arg 3940 3945 3950 Leu Asp Val Glu Phe Lys Pro Leu Ala Pro Asp Gly Val Leu Leu Phe 3955 3960 3965 Ser Gly Gly Lys Ser Gly Pro Val Glu Asp Phe Val Ser Leu Ala Met 3970 3975 3980 Val Gly Gly His Leu Glu Phe Arg Tyr Glu Leu Gly Ser Gly Leu Ala 3985 3990 3995 4000 Val Leu Arg Ser Ala Glu Pro Leu Ala Leu Gly Arg Trp His Arg Val 4005 4010 4015 Ser Ala Glu Arg Leu Asn Lys Asp Gly Ser Leu Arg Val Asn Gly Gly 4020 4025 4030 Arg Pro Val Leu Arg Ser Ser Pro Gly Lys Ser Gln Gly Leu Asn Leu 4035 4040 4045 His Thr Leu Leu Tyr Leu Gly Gly Val Glu Pro Ser Val Pro Leu Ser 4050 4055 4060 Pro Ala Thr Asn Met Ser Ala His Phe Arg Gly Cys Val Gly Glu Val 4065 4070 4075 4080 Ser Val Asn Gly Lys Arg Leu Asp Leu Thr Tyr Ser Phe Leu Gly Ser 4085 4090 4095 Gln Gly Ile Gly Gln Cys Tyr Asp Ser Ser Pro Cys Glu Arg Gln Pro 4100 4105 4110 Cys Gln His Gly Ala Thr Cys Met Pro Ala Gly Glu Tyr Glu Phe Gln 4115 4120 4125 Cys Leu Cys Arg Asp Gly Phe Lys Gly Asp Leu Cys Glu His Glu Glu 4130 4135 4140 Asn Pro Cys Gln Leu Arg Glu Pro Cys Leu His Gly Gly Thr Cys Gln 4145 4150 4155 4160 Gly Thr Arg Cys Leu Cys Leu Pro Gly Phe Ser Gly Pro Arg Cys Gln 4165 4170 4175 Gln Gly Ser Gly His Gly Ile Ala Glu Ser Asp Trp His Leu Glu Gly 4180 4185 4190 Ser Gly Gly Asn Asp Ala Pro Gly Gln Tyr Gly Ala Tyr Phe His Asp 4195 4200 4205 Asp Gly Phe Leu Ala Phe Pro Gly His Val Phe Ser Arg Ser Leu Pro 4210 4215 4220 Glu Val Pro Glu Thr Ile Glu Leu Glu Val Arg Thr Ser Thr Ala Ser 4225 4230 4235 4240 Gly Leu Leu Leu Trp Gln Gly Val Glu Val Gly Glu Ala Gly Gln Gly 4245 4250 4255 Lys Asp Phe Ile Ser Leu Gly Leu Gln Asp Gly His Leu Val Phe Arg 4260 4265 4270 Tyr Gln Leu Gly Ser Gly Glu Ala Arg Leu Val Ser Glu Asp Pro Ile 4275 4280 4285 Asn Asp Gly Glu Trp His Arg Val Thr Ala Leu Arg Glu Gly Arg Arg 4290 4295 4300 Gly Ser Ile Gln Val Asp Gly Glu Glu Leu Val Ser Gly Arg Ser Pro 4305 4310 4315 4320 Gly Pro Asn Val Ala Val Asn Ala Lys Gly Ser Val Tyr Ile Gly Gly 4325 4330 4335 Ala Pro Asp Val Ala Thr Leu Thr Gly Gly Arg Phe Ser Ser Gly Ile 4340 4345 4350 Thr Gly Cys Val Lys Asn Leu Val Leu His Ser Ala Arg Pro Gly Ala 4355 4360 4365 Pro Pro Pro Gln Pro Leu Asp Leu Gln His Arg Ala Gln Ala Gly Ala 4370 4375 4380 Asn Thr Arg Pro Cys Pro Ser 4385 4390 3 705 PRT human 3 Glu Ile Lys Ile Thr Phe Arg Pro Asp Ser Ala Asp Gly Met Leu Leu 1 5 10 15 Tyr Asn Gly Gln Lys Arg Val Pro Gly Ser Pro Thr Asn Leu Ala Asn 20 25 30 Arg Gln Pro Asp Phe Ile Ser Phe Gly Leu Val Gly Gly Arg Pro Glu 35 40 45 Phe Arg Phe Asp Ala Gly Ser Gly Met Ala Thr Ile Arg His Pro Thr 50 55 60 Pro Leu Ala Leu Gly His Phe His Thr Val Thr Leu Leu Arg Ser Leu 65 70 75 80 Thr Gln Gly Ser Leu Ile Val Gly Asp Leu Ala Pro Val Asn Gly Thr 85 90 95 Ser Gln Gly Lys Phe Gln Gly Leu Asp Leu Asn Glu Glu Leu Tyr Leu 100 105 110 Gly Gly Tyr Pro Asp Tyr Gly Ala Ile Pro Lys Ala Gly Leu Ser Ser 115 120 125 Gly Phe Ile Gly Cys Val Arg Glu Leu Arg Ile Gln Gly Glu Glu Ile 130 135 140 Val Phe His Asp Leu Asn Leu Thr Ala His Gly Ile Ser His Cys Pro 145 150 155 160 Thr Cys Arg Asp Arg Pro Cys Gln Asn Gly Gly Gln Cys His Asp Ser 165 170 175 Glu Ser Ser Ser Tyr Val Cys Val Cys Pro Ala Gly Phe Thr Gly Ser 180 185 190 Arg Cys Glu His Ser Gln Ala Leu His Cys His Pro Glu Ala Cys Gly 195 200 205 Pro Asp Ala Thr Cys Val Asn Arg Pro Asp Gly Arg Gly Tyr Thr Cys 210 215 220 Arg Cys His Leu Gly Arg Ser Gly Leu Arg Cys Glu Glu Gly Val Thr 225 230 235 240 Val Thr Thr Pro Ser Leu Ser Gly Ala Gly Ser Tyr Leu Ala Leu Pro 245 250 255 Ala Leu Thr Asn Thr His His Glu Leu Arg Leu Asp Val Glu Phe Lys 260 265 270 Pro Leu Ala Pro Asp Gly Val Leu Leu Phe Ser Gly Gly Lys Ser Gly 275 280 285 Pro Val Glu Asp Phe Val Ser Leu Ala Met Val Gly Gly His Leu Glu 290 295 300 Phe Arg Tyr Glu Leu Gly Ser Gly Leu Ala Val Leu Arg Ser Ala Glu 305 310 315 320 Pro Leu Ala Leu Gly Arg Trp His Arg Val Ser Ala Glu Arg Leu Asn 325 330 335 Lys Asp Gly Ser Leu Arg Val Asn Gly Gly Arg Pro Val Leu Arg Ser 340 345 350 Ser Pro Gly Lys Ser Gln Gly Leu Asn Leu His Thr Leu Leu Tyr Leu 355 360 365 Gly Gly Val Glu Pro Ser Val Pro Leu Ser Pro Ala Thr Asn Met Ser 370 375 380 Ala His Phe Arg Gly Cys Val Gly Glu Val Ser Val Asn Gly Lys Arg 385 390 395 400 Leu Asp Leu Thr Tyr Ser Phe Leu Gly Ser Gln Gly Ile Gly Gln Cys 405 410 415 Tyr Asp Ser Ser Pro Cys Glu Arg Gln Pro Cys Gln His Gly Ala Thr 420 425 430 Cys Met Pro Ala Gly Glu Tyr Glu Phe Gln Cys Leu Cys Arg Asp Gly 435 440 445 Phe Lys Gly Asp Leu Cys Glu His Glu Glu Asn Pro Cys Gln Leu Arg 450 455 460 Glu Pro Cys Leu His Gly Gly Thr Cys Gln Gly Thr Arg Cys Leu Cys 465 470 475 480 Leu Pro Gly Phe Ser Gly Pro Arg Cys Gln Gln Gly Ser Gly His Gly 485 490 495 Ile Ala Glu Ser Asp Trp His Leu Glu Gly Ser Gly Gly Asn Asp Ala 500 505 510 Pro Gly Gln Tyr Gly Ala Tyr Phe His Asp Asp Gly Phe Leu Ala Phe 515 520 525 Pro Gly His Val Phe Ser Arg Ser Leu Pro Glu Val Pro Glu Thr Ile 530 535 540 Glu Leu Glu Val Arg Thr Ser Thr Ala Ser Gly Leu Leu Leu Trp Gln 545 550 555 560 Gly Val Glu Val Gly Glu Ala Gly Gln Gly Lys Asp Phe Ile Ser Leu 565 570 575 Gly Leu Gln Asp Gly His Leu Val Phe Arg Tyr Gln Leu Gly Ser Gly 580 585 590 Glu Ala Arg Leu Val Ser Glu Asp Pro Ile Asn Asp Gly Glu Trp His 595 600 605 Arg Val Thr Ala Leu Arg Glu Gly Arg Arg Gly Ser Ile Gln Val Asp 610 615 620 Gly Glu Glu Leu Val Ser Gly Arg Ser Pro Gly Pro Asn Val Ala Val 625 630 635 640 Asn Ala Lys Gly Ser Val Tyr Ile Gly Gly Ala Pro Asp Val Ala Thr 645 650 655 Leu Thr Gly Gly Arg Phe Ser Ser Gly Ile Thr Gly Cys Val Lys Asn 660 665 670 Leu Val Leu His Ser Ala Arg Pro Gly Ala Pro Pro Pro Gln Pro Leu 675 680 685 Asp Leu Gln His Arg Ala Gln Ala Gly Ala Asn Thr Arg Pro Cys Pro 690 695 700 Ser 705 4 495 PRT human 4 Glu Ile Lys Ile Thr Phe Arg Pro Asp Ser Ala Asp Gly Met Leu Leu 1 5 10 15 Tyr Asn Gly Gln Lys Arg Val Pro Gly Ser Pro Thr Asn Leu Ala Asn 20 25 30 Arg Gln Pro Asp Phe Ile Ser Phe Gly Leu Val Gly Gly Arg Pro Glu 35 40 45 Phe Arg Phe Asp Ala Gly Ser Gly Met Ala Thr Ile Arg His Pro Thr 50 55 60 Pro Leu Ala Leu Gly His Phe His Thr Val Thr Leu Leu Arg Ser Leu 65 70 75 80 Thr Gln Gly Ser Leu Ile Val Gly Asp Leu Ala Pro Val Asn Gly Thr 85 90 95 Ser Gln Gly Lys Phe Gln Gly Leu Asp Leu Asn Glu Glu Leu Tyr Leu 100 105 110 Gly Gly Tyr Pro Asp Tyr Gly Ala Ile Pro Lys Ala Gly Leu Ser Ser 115 120 125 Gly Phe Ile Gly Cys Val Arg Glu Leu Arg Ile Gln Gly Glu Glu Ile 130 135 140 Val Phe His Asp Leu Asn Leu Thr Ala His Gly Ile Ser His Cys Pro 145 150 155 160 Thr Cys Arg Asp Arg Pro Cys Gln Asn Gly Gly Gln Cys His Asp Ser 165 170 175 Glu Ser Ser Ser Tyr Val Cys Val Cys Pro Ala Gly Phe Thr Gly Ser 180 185 190 Arg Cys Glu His Ser Gln Ala Leu His Cys His Pro Glu Ala Cys Gly 195 200 205 Pro Asp Ala Thr Cys Val Asn Arg Pro Asp Gly Arg Gly Tyr Thr Cys 210 215 220 Arg Cys His Leu Gly Arg Ser Gly Leu Arg Cys Glu Glu Gly Val Thr 225 230 235 240 Val Thr Thr Pro Ser Leu Ser Gly Ala Gly Ser Tyr Leu Ala Leu Pro 245 250 255 Ala Leu Thr Asn Thr His His Glu Leu Arg Leu Asp Val Glu Phe Lys 260 265 270 Pro Leu Ala Pro Asp Gly Val Leu Leu Phe Ser Gly Gly Lys Ser Gly 275 280 285 Pro Val Glu Asp Phe Val Ser Leu Ala Met Val Gly Gly His Leu Glu 290 295 300 Phe Arg Tyr Glu Leu Gly Ser Gly Leu Ala Val Leu Arg Ser Ala Glu 305 310 315 320 Pro Leu Ala Leu Gly Arg Trp His Arg Val Ser Ala Glu Arg Leu Asn 325 330 335 Lys Asp Gly Ser Leu Arg Val Asn Gly Gly Arg Pro Val Leu Arg Ser 340 345 350 Ser Pro Gly Lys Ser Gln Gly Leu Asn Leu His Thr Leu Leu Tyr Leu 355 360 365 Gly Gly Val Glu Pro Ser Val Pro Leu Ser Pro Ala Thr Asn Met Ser 370 375 380 Ala His Phe Arg Gly Cys Val Gly Glu Val Ser Val Asn Gly Lys Arg 385 390 395 400 Leu Asp Leu Thr Tyr Ser Phe Leu Gly Ser Gln Gly Ile Gly Gln Cys 405 410 415 Tyr Asp Ser Ser Pro Cys Glu Arg Gln Pro Cys Gln His Gly Ala Thr 420 425 430 Cys Met Pro Ala Gly Glu Tyr Glu Phe Gln Cys Leu Cys Arg Asp Gly 435 440 445 Phe Lys Gly Asp Leu Cys Glu His Glu Glu Asn Pro Cys Gln Leu Arg 450 455 460 Glu Pro Cys Leu His Gly Gly Thr Cys Gln Gly Thr Arg Cys Leu Cys 465 470 475 480 Leu Pro Gly Phe Ser Gly Pro Arg Cys Gln Gln Gly Ser Gly His 485 490 495 5 240 PRT human 5 Glu Ile Lys Ile Thr Phe Arg Pro Asp Ser Ala Asp Gly Met Leu Leu 1 5 10 15 Tyr Asn Gly Gln Lys Arg Val Pro Gly Ser Pro Thr Asn Leu Ala Asn 20 25 30 Arg Gln Pro Asp Phe Ile Ser Phe Gly Leu Val Gly Gly Arg Pro Glu 35 40 45 Phe Arg Phe Asp Ala Gly Ser Gly Met Ala Thr Ile Arg His Pro Thr 50 55 60 Pro Leu Ala Leu Gly His Phe His Thr Val Thr Leu Leu Arg Ser Leu 65 70 75 80 Thr Gln Gly Ser Leu Ile Val Gly Asp Leu Ala Pro Val Asn Gly Thr 85 90 95 Ser Gln Gly Lys Phe Gln Gly Leu Asp Leu Asn Glu Glu Leu Tyr Leu 100 105 110 Gly Gly Tyr Pro Asp Tyr Gly Ala Ile Pro Lys Ala Gly Leu Ser Ser 115 120 125 Gly Phe Ile Gly Cys Val Arg Glu Leu Arg Ile Gln Gly Glu Glu Ile 130 135 140 Val Phe His Asp Leu Asn Leu Thr Ala His Gly Ile Ser His Cys Pro 145 150 155 160 Thr Cys Arg Asp Arg Pro Cys Gln Asn Gly Gly Gln Cys His Asp Ser 165 170 175 Glu Ser Ser Ser Tyr Val Cys Val Cys Pro Ala Gly Phe Thr Gly Ser 180 185 190 Arg Cys Glu His Ser Gln Ala Leu His Cys His Pro Glu Ala Cys Gly 195 200 205 Pro Asp Ala Thr Cys Val Asn Arg Pro Asp Gly Arg Gly Tyr Thr Cys 210 215 220 Arg Cys His Leu Gly Arg Ser Gly Leu Arg Cys Glu Glu Gly Val Thr 225 230 235 240 6 161 PRT human 6 Glu Ile Lys Ile Thr Phe Arg Pro Asp Ser Ala Asp Gly Met Leu Leu 1 5 10 15 Tyr Asn Gly Gln Lys Arg Val Pro Gly Ser Pro Thr Asn Leu Ala Asn 20 25 30 Arg Gln Pro Asp Phe Ile Ser Phe Gly Leu Val Gly Gly Arg Pro Glu 35 40 45 Phe Arg Phe Asp Ala Gly Ser Gly Met Ala Thr Ile Arg His Pro Thr 50 55 60 Pro Leu Ala Leu Gly His Phe His Thr Val Thr Leu Leu Arg Ser Leu 65 70 75 80 Thr Gln Gly Ser Leu Ile Val Gly Asp Leu Ala Pro Val Asn Gly Thr 85 90 95 Ser Gln Gly Lys Phe Gln Gly Leu Asp Leu Asn Glu Glu Leu Tyr Leu 100 105 110 Gly Gly Tyr Pro Asp Tyr Gly Ala Ile Pro Lys Ala Gly Leu Ser Ser 115 120 125 Gly Phe Ile Gly Cys Val Arg Glu Leu Arg Ile Gln Gly Glu Glu Ile 130 135 140 Val Phe His Asp Leu Asn Leu Thr Ala His Gly Ile Ser His Cys Pro 145 150 155 160 Thr 7 79 PRT human 7 Cys Arg Asp Arg Pro Cys Gln Asn Gly Gly Gln Cys His Asp Ser Glu 1 5 10 15 Ser Ser Ser Tyr Val Cys Val Cys Pro Ala Gly Phe Thr Gly Ser Arg 20 25 30 Cys Glu His Ser Gln Ala Leu His Cys His Pro Glu Ala Cys Gly Pro 35 40 45 Asp Ala Thr Cys Val Asn Arg Pro Asp Gly Arg Gly Tyr Thr Cys Arg 50 55 60 Cys His Leu Gly Arg Ser Gly Leu Arg Cys Glu Glu Gly Val Thr 65 70 75 8 255 PRT human 8 Val Thr Thr Pro Ser Leu Ser Gly Ala Gly Ser Tyr Leu Ala Leu Pro 1 5 10 15 Ala Leu Thr Asn Thr His His Glu Leu Arg Leu Asp Val Glu Phe Lys 20 25 30 Pro Leu Ala Pro Asp Gly Val Leu Leu Phe Ser Gly Gly Lys Ser Gly 35 40 45 ] Pro Val Glu Asp Phe Val Ser Leu Ala Met Val Gly Gly His Leu Glu 50 55 60 Phe Arg Tyr Glu Leu Gly Ser Gly Leu Ala Val Leu Arg Ser Ala Glu 65 70 75 80 Pro Leu Ala Leu Gly Arg Trp His Arg Val Ser Ala Glu Arg Leu Asn 85 90 95 Lys Asp Gly Ser Leu Arg Val Asn Gly Gly Arg Pro Val Leu Arg Ser 100 105 110 Ser Pro Gly Lys Ser Gln Gly Leu Asn Leu His Thr Leu Leu Tyr Leu 115 120 125 Gly Gly Val Glu Pro Ser Val Pro Leu Ser Pro Ala Thr Asn Met Ser 130 135 140 Ala His Phe Arg Gly Cys Val Gly Glu Val Ser Val Asn Gly Lys Arg 145 150 155 160 Leu Asp Leu Thr Tyr Ser Phe Leu Gly Ser Gln Gly Ile Gly Gln Cys 165 170 175 Tyr Asp Ser Ser Pro Cys Glu Arg Gln Pro Cys Gln His Gly Ala Thr 180 185 190 Cys Met Pro Ala Gly Glu Tyr Glu Phe Gln Cys Leu Cys Arg Asp Gly 195 200 205 Phe Lys Gly Asp Leu Cys Glu His Glu Glu Asn Pro Cys Gln Leu Arg 210 215 220 Glu Pro Cys Leu His Gly Gly Thr Cys Gln Gly Thr Arg Cys Leu Cys 225 230 235 240 Leu Pro Gly Phe Ser Gly Pro Arg Cys Gln Gln Gly Ser Gly His 245 250 255 9 284 PRT human 9 Cys Glu Arg Gln Pro Cys Gln His Gly Ala Thr Cys Met Pro Ala Gly 1 5 10 15 Glu Tyr Glu Phe Gln Cys Leu Cys Arg Asp Gly Phe Lys Gly Asp Leu 20 25 30 Cys Glu His Glu Glu Asn Pro Cys Gln Leu Arg Glu Pro Cys Leu His 35 40 45 Gly Gly Thr Cys Gln Gly Thr Arg Cys Leu Cys Leu Pro Gly Phe Ser 50 55 60 Gly Pro Arg Cys Gln Gln Gly Ser Gly His Gly Ile Ala Glu Ser Asp 65 70 75 80 Trp His Leu Glu Gly Ser Gly Gly Asn Asp Ala Pro Gly Gln Tyr Gly 85 90 95 Ala Tyr Phe His Asp Asp Gly Phe Leu Ala Phe Pro Gly His Val Phe 100 105 110 Ser Arg Ser Leu Pro Glu Val Pro Glu Thr Ile Glu Leu Glu Val Arg 115 120 125 Thr Ser Thr Ala Ser Gly Leu Leu Leu Trp Gln Gly Val Glu Val Gly 130 135 140 Glu Ala Gly Gln Gly Lys Asp Phe Ile Ser Leu Gly Leu Gln Asp Gly 145 150 155 160 His Leu Val Phe Arg Tyr Gln Leu Gly Ser Gly Glu Ala Arg Leu Val 165 170 175 Ser Glu Asp Pro Ile Asn Asp Gly Glu Trp His Arg Val Thr Ala Leu 180 185 190 Arg Glu Gly Arg Arg Gly Ser Ile Gln Val Asp Gly Glu Glu Leu Val 195 200 205 Ser Gly Arg Ser Pro Gly Pro Asn Val Ala Val Asn Ala Lys Gly Ser 210 215 220 Val Tyr Ile Gly Gly Ala Pro Asp Val Ala Thr Leu Thr Gly Gly Arg 225 230 235 240 Phe Ser Ser Gly Ile Thr Gly Cys Val Lys Asn Leu Val Leu His Ser 245 250 255 Ala Arg Pro Gly Ala Pro Pro Pro Gln Pro Leu Asp Leu Gln His Arg 260 265 270 Ala Gln Ala Gly Ala Asn Thr Arg Pro Cys Pro Ser 275 280 10 210 PRT human 10 Gly Ile Ala Glu Ser Asp Trp His Leu Glu Gly Ser Gly Gly Asn Asp 1 5 10 15 Ala Pro Gly Gln Tyr Gly Ala Tyr Phe His Asp Asp Gly Phe Leu Ala 20 25 30 Phe Pro Gly His Val Phe Ser Arg Ser Leu Pro Glu Val Pro Glu Thr 35 40 45 Ile Glu Leu Glu Val Arg Thr Ser Thr Ala Ser Gly Leu Leu Leu Trp 50 55 60 Gln Gly Val Glu Val Gly Glu Ala Gly Gln Gly Lys Asp Phe Ile Ser 65 70 75 80 Leu Gly Leu Gln Asp Gly His Leu Val Phe Arg Tyr Gln Leu Gly Ser 85 90 95 Gly Glu Ala Arg Leu Val Ser Glu Asp Pro Ile Asn Asp Gly Glu Trp 100 105 110 His Arg Val Thr Ala Leu Arg Glu Gly Arg Arg Gly Ser Ile Gln Val 115 120 125 Asp Gly Glu Glu Leu Val Ser Gly Arg Ser Pro Gly Pro Asn Val Ala 130 135 140 Val Asn Ala Lys Gly Ser Val Tyr Ile Gly Gly Ala Pro Asp Val Ala 145 150 155 160 Thr Leu Thr Gly Gly Arg Phe Ser Ser Gly Ile Thr Gly Cys Val Lys 165 170 175 Asn Leu Val Leu His Ser Ala Arg Pro Gly Ala Pro Pro Pro Gln Pro 180 185 190 Leu Asp Leu Gln His Arg Ala Gln Ala Gly Ala Asn Thr Arg Pro Cys 195 200 205 Pro Ser 210 

What is claimed is:
 1. A pharmaceutical composition, comprising: a) a polypeptide consisting of the amino acid sequence SEQ ID NO:3 or a fragment of said polypeptide which comprises the amino acid SEQ ID NO:10 and has agiogenesis-inhibiting activity; and b) a pharmaceutically acceptable carrier or excipient.
 2. The pharmaceutical composition of claim 1, wherein said fragment consists of the amino acid sequence SEQ ID NO:9.
 3. The pharmaceutical composition of claim 1, wherein said fragment consists of the amino acid sequence SEQ ID NO:10. 